cDNA Synthesis Improvements

Abstract

The present invention generally relates to methods of making cDNA molecules and cDNA libraries. The invention also relates to cDNA molecules and cDNA libraries produced according to these methods, as well as to vectors and host cells containing such cDNA molecules and libraries. The invention also relates to kits for making the cDNA molecules and libraries of the invention.

1 . A method for synthesizing one or more cDNA molecules or population of cDNA molecules comprising:
 mixing at least one mRNA or poly A RNA template or population of such templates with at least one polypeptide having reverse transcriptase activity; and   incubating said mixture under conditions sufficient to increase the amount or percentage of full-length cDNA molecules synthesized.   
     
     
         2 . The method of  claim 1 , wherein said conditions reduce or substantially reduce internal priming. 
     
     
         3 . The method of  claim 1 , wherein said polypeptide is a reverse transcriptase selected from the group consisting of M-MLV RT, RSV RT, AMV RT, RA V RT, MA V RT, and HIV RT, and derivatives, fragments, mutations and variants thereof. 
     
     
         4 . The method of  claim 3 , wherein said reverse transcriptase is reduced or substantially reduced in RNase H activity. 
     
     
         5 . The method of  claim 2 , wherein said conditions comprise annealing or hybridizing one or more primers to said templates at elevated temperatures. 
     
     
         6 . The method of  claim 5 , wherein said elevated temperature ranges from about 20° C. to about 90° C. 
     
     
         7 . The method of  claim 2 , wherein said conditions comprise lowering the amount of primer relative to the amount of said template. 
     
     
         8 . The method of  claim 7 , wherein the ratio of said primer to said template ranges from about 5:1 to about 1:20. 
     
     
         9 . The method of  claim 1 , wherein said conditions comprise the use of an inhibitor of the polypeptide having reverse transcriptase activity. 
     
     
         10 . The method of  claim 9 , wherein said inhibitor is an antibody or antibody fragment. 
     
     
         11 . The method of  claim 10 , wherein said antibody or antibody fragment is polyclonal or monoclonal. 
     
     
         12 . The method of  claim 2 , wherein said conditions comprise the use of a primer having a high specificity. 
     
     
         13 . The method of  claim 2 , wherein said conditions comprise increasing the length of said primer. 
     
     
         14 . The method of  claim 13 , wherein the length of said primer which hybridizes to said template ranges from about 20 bases to about 60 bases. 
     
     
         15 . The method of  claim 1 , wherein said method further comprises incubating at least one of said cDNA molecules under conditions sufficient to make at least one second nucleic acid molecule complementary to all or a portion of said at least one cDNA molecule, thereby producing one or more double stranded cDNA molecules. 
     
     
         16 . The method of  claim 15 , wherein said conditions for making said second nucleic acid molecule increases the amount or percentage of full-length double stranded cDNA molecules. 
     
     
         17 .- 28 . (canceled) 
     
     
         29 . A composition for making an increased amount or percentage of full-length cDNA comprising at least one component selected from the group consisting of one or more primers, one or more reverse transcription inhibitors, one or more reverse transcription enzymes, one or more nucleotides, one or more cap binding molecules, one or more reverse transcription buffers. 
     
     
         30 .- 32 . (canceled) 
     
     
         33 . The composition of  claim 29 , wherein said one or more reverse transcriptase inhibitors is an antibody or antibody fragment.